JP5881599B2 - Rapid sterile microassay - Google Patents

Description

  This application claims the benefit of US Provisional Application No. 61 / 175,271, filed May 4, 2009. The entire teachings of the above application are incorporated herein by reference.

  Federal regulations in the United States and similar regulations in other countries require sterility testing to ensure that the pharmaceutical product is substantially free of microorganisms such as bacteria and fungi. Such sterility testing is performed using three common methods: direct transfer sterility testing, membrane filtration sterility testing and product flash sterility testing. Traditional sterility tests include two liquid nutrient media (tryptic soy broth (TBS) incubated at 20 ° C. to 25 ° C. and thioglycolic acid nutrient medium (FTM) incubated at 30 ° C. to 35 ° C.), and rinse Performed with fluid and requires an incubation time of 14 days (eg, USP <71> “Sterility Tests”, Pharmacopeical Forum. USP30-NF25 to First Supplement The United States, Inc., Conv. 6 “Sterility” (European Pharmacopoiea 2.1.6 “Sterility” EP 6th Edition (2007)) and See other related Pharmacopoeia). Liquid thioglycolic acid medium (FTM) contains two phases-the lower phase of the liquid medium is an anaerobic phase and the upper phase contains oxygen for aerobic incubation.

  In general, liquid, emulsified or dissolved pharmaceutical or medical products (eg active ingredients, parenteral formulations, eye drops, intranasal sprays) to test the sterility of a pharmaceutical product using membrane filtration Etc.) is filtered through a 0.45 micron membrane and the membrane is transferred to the appropriate test medium (FTM and TSB) and incubated for 14 days. If the microorganism grows in culture, the pharmaceutical product is not sterile and samples can be taken for microbiological identification. It takes a long time for incubation and also for microbiological identification of organisms growing in culture, which is disadvantageous and reduces the availability of pharmaceutical products to patients in need thereof. Can be limited. For example, in the case of a medical crisis, current sterility testing that requires a 14-day incubation period can delay the availability of drugs or vaccines that can suppress or end the crisis. Therefore, there is a need for a rapid sterility test method.

  The present invention provides: a) providing a filterable pharmaceutical composition; b) filtering the pharmaceutical composition to provide at least three filter membranes on which the pharmaceutical composition has been deposited. C) placing the at least three filter membranes in a solid medium to produce at least three filter cultures; d) removing the filter cultures from about 13 days. I) at least one filtered culture at 20 ° C. to 25 ° C. under aerobic conditions; ii) at least one filtered culture at 30 ° C. Culturing at 30 ° C. to 35 ° C. under anaerobic conditions; and e) surviving in the membrane; Detecting a biological cell, microcolony or colony, wherein the presence of viable microbial cells, microcolonies or colonies in the membrane indicates the presence of viable microorganisms in the pharmaceutical composition Relates to a method for detecting viable microorganisms.

  The method may further comprise the step of filtering the wash solution after filtering the pharmaceutical composition.

  In some embodiments, the membrane is a polyvinylidene fluoride membrane, a glass fiber membrane, a polycarbonate membrane, a polyethylene terephthalate membrane, a mixed cellulose ester (cellulose acetate and cellulose nitrate), a phosphocellulose membrane, a DEAE membrane, a nylon mesh membrane, Alternatively, it is a polytetrafluoroethylene membrane. Preferably, the membrane has a pore size of about 0.45 μm.

  The solid medium was FTM-A (liquid thioglycolic acid medium (final concentration) containing 1.075% agar), BHI (Brain Heart Infusion Agar), Difco brewer anaerobic agar, R2A agar, Schaedler. It can be selected from the group consisting of blood agar, Caso-agar ICR (tryptic soy agar), Columbia agar 5% blood, and CDC anaerobic blood agar.

  In some embodiments, the method can include culturing the filtrate culture for a period of about 2 to about 7 days.

  In some embodiments, viable microbial cells, microcolonies, or colonies are luminescent, such as a bioluminescent assay that detects adenosine triphosphate (ATP) produced by viable microbial cells, microcolonies, or colonies in the membrane. Detect using assay. The luminescence assay can include a luciferase assay.

  In some embodiments, the luminescence assay detects a nucleic acid hybridization product formed between the probe and the nucleic acid that is endogenous to the microorganism. The luminescence assay can include a peroxidase reaction.

  In some embodiments, the luminescence can be detected using a charge coupled device camera and image analysis software. In some embodiments, the number of viable microbial cells, viable microbial microcolonies, or viable microbial colonies may be quantified or measured.

  In some embodiments, the pharmaceutical composition is a liquid composition. The liquid composition can be a parenteral composition, an oral composition, a nasal composition, an ophthalmic composition, or a vaccine.

The invention also provides: a) providing a filterable pharmaceutical composition; b) filtering the pharmaceutical composition to provide at least three filter membranes on which the pharmaceutical composition filter cake has been deposited; c) placing at least three filter membranes in solid medium to produce at least three filtrate cultures; d) i. provided that the filter cultures are not cultured for longer than about 13 days. A) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions; ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and iii) at least one Culturing two filter cake cultures at 30-35 ° C. under anaerobic conditions; and e) detecting adenosine triphosphate (ATP) in the membrane, wherein The presence of ATP in Nburan includes the steps of indicating the presence of viable microorganisms in the pharmaceutical composition, including, relates to a method for detecting viable microorganisms in the pharmaceutical composition.
The present application provides, for example, the following items in one embodiment:
(Item 1)
A method for detecting viable microorganisms in a pharmaceutical composition comprising:
a) providing a filterable pharmaceutical composition;
b) filtering the pharmaceutical composition to provide at least three filter membranes on which the filter cake of the pharmaceutical composition is deposited;
c) placing the at least three filter membranes in a solid medium to produce at least three filtrate cultures;
d)
i) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions;
ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and
iii) at least one filter cake culture at 30 ° C. to 35 ° C. under anaerobic conditions,
Culturing, wherein none of the filter cake cultures are cultured for a period longer than about 13 days; and
e) detecting viable microbial cells, microcolonies or colonies in the membrane, wherein the presence of viable microbial cells, microcolony or colonies in the membrane indicates the presence of viable microorganisms in the pharmaceutical composition , Including methods.
(Item 2)
Item 2. The method according to Item 1, wherein step b) further comprises a step of filtering the washing solution after filtering the pharmaceutical composition.
(Item 3)
The membrane is a polyvinylidene fluoride membrane, glass fiber membrane, polycarbonate membrane, polyethylene terephthalate membrane, mixed cellulose ester (cellulose acetate and cellulose nitrate), phosphocellulose membrane, DEAE membrane, nylon mesh membrane, or polytetrafluoroethylene (polytetrafluoroethylene). 3. The method according to item 1 or 2, which is a membrane.
(Item 4)
The method of any one of the preceding items, wherein the membrane has a pore size of about 0.45 μm.
(Item 5)
The solid medium is FTM-A (liquid thioglycolic acid medium (final concentration) containing 1.075% agar), BHI (brain heart infusion agar medium), Difco brewer anaerobic agar medium, R2A agar medium. Any one of the preceding items selected from the group consisting of: Schadler blood agar medium, Caso-agar medium ICR (tryptic soy agar medium), Columbia agar medium 5% blood, and CDC anaerobic blood agar medium. the method of.
(Item 6)
The method according to any one of the preceding items, wherein, in step d), the filtered culture is cultured for a period sufficient to produce a detectable amount of ATP.
(Item 7)
Item 7. The method according to Item 6, wherein the filtered culture is cultured for a period of about 2 days to about 7 days.
(Item 8)
The method according to any one of the preceding items, wherein in step e) viable microbial cells, microcolony or colonies are detected using a luminescence assay.
(Item 9)
9. The method of item 8, wherein the luminescent assay detects viable microbial cells, microcolony, or adenosine triphosphate (ATP) produced by the membrane in the membrane.
(Item 10)
9. The method of item 8, wherein the luminescence assay comprises a luciferase assay.
(Item 11)
9. The method of item 8, wherein the luminescence assay detects a nucleic acid hybridization product formed between a probe and a nucleic acid that is endogenous to the microorganism.
(Item 12)
Item 12. The method according to Item 11, wherein the luminescence assay comprises a peroxidase reaction.
(Item 13)
13. The method according to any one of items 8-12, wherein luminescence is detected using a charge coupled device camera and image analysis software.
(Item 14)
14. The method according to any one of items 8 to 13, wherein the viable microbial cell, viable microbial microcolony, or viable microbial colony is counted.
(Item 15)
The method according to any one of the preceding items, wherein the pharmaceutical composition is a liquid composition.
(Item 16)
Item 16. The method according to Item 15, wherein the liquid composition is a parenteral composition, an oral composition, a nasal composition, or an ophthalmic composition.
(Item 17)
Item 16. The method according to Item 15, wherein the liquid composition is a vaccine.
(Item 18)
The vaccine is an anthrax vaccine; tuberculosis vaccine; borreliosis vaccine; diphtheria toxoid and tetanus toxoid vaccine; diphtheria toxoid and tetanus toxoid and pertussis vaccine; diphtheria toxoid and tetanus toxoid and acellular pertussis vaccine; Acellular pertussis and Haemophilus influenzae type b conjugate vaccine; Diphtheria and tetanus toxoid and acellular pertussis and Haemophilus influenzae type b conjugate and poliovirus inactivated vaccine; Hepatitis A virus vaccine; Hepatitis A virus and Hepatitis B virus Vaccine; Hepatitis B virus vaccine; Helicobacter pylori vaccine; Haemophilus influenzae type b vaccine; Influenza virus vaccine Poliovirus vaccine; Neisseria meningitidis vaccine; measles virus, mumps virus, rubella virus vaccine; measles virus, mumps virus, rubella virus and chickenpox Viral vaccines; Streptococcus pneumoniae vaccine; Rabies vaccine; Respiratory syncytial virus vaccine; Smallpox vaccine; Toxoplasmosis (toxoplasm gondii) vaccine; 18. The method according to item 17, wherein the method is selected from the group consisting of (bacteria tuberculosis) vaccine; and varicella (varicella, varicella-zoster virus) vaccine.
(Item 19)
18. The method according to item 17, wherein the vaccine is a bird flu vaccine or a swine flu vaccine.
(Item 20)
A method for detecting viable microorganisms in a pharmaceutical composition comprising:
a) providing a filterable pharmaceutical composition;
b) filtering the pharmaceutical composition to provide at least three membranes on which the filter cake of the pharmaceutical composition is deposited;
c) placing the at least three filters / membranes in a solid medium to produce at least three filtrate cultures;
d)
i) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions;
ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and
iii) at least one filter cake culture at 30 ° C. to 35 ° C. under anaerobic conditions,
Culturing, wherein none of the filter cake cultures are cultured for a period longer than about 13 days; and
e) detecting adenosine triphosphate (ATP) in the membrane, wherein the presence of ATP in the membrane indicates the presence of viable microorganisms in the pharmaceutical composition.
(Item 21)
A sterile pharmaceutical composition that is screened for viable microorganisms and sterility is confirmed using the method according to any one of the preceding items.

FIG. 1 shows M.M. over a 10 minute time frame. It is a graph which shows the result of UV processing of osloensis. The number of colony forming units (CFU) present after 4 days of incubation is shown. The graph shows more than 50% reduction in CFU after 4 minutes (3 runs). FIG. 2 shows E.I. It is a graph which shows the result of the heat processing of E. coli. The number of colony forming units (CFU) after 3 days incubation is shown. The graph shows a 50% or greater reduction in CFU after 3 minutes (3 runs). FIG. 3 is a graph showing the results of parenteral formulation treatment over a 10 minute time frame. The number of colony forming units (CFU) after 5 days incubation is shown. The graph shows more than 50% reduction in CFU after 2 minutes (3 runs). FIG. 4A shows the M.P. heat treatment for 3 minutes at 70.degree. It is a microscope picture of luteus cell. FIG. 4B shows untreated M. pylori after 3 days incubation at 30 ° C. to 35 ° C. on tryptic soy agar medium. It is a microscope picture of luteus cell. FIG. E. coli culture, heat treatment E. coli, UV treatment E. coli. E. coli and Voltaren treatment It is a graph which shows the absorbance data of E. coli. FIG. E. coli culture, heat treatment E. coli, UV treatment E. coli. E. coli and Voltaren treatment It is a graph which shows the absorbance data of E. coli. FIG. E. coli growth curve and heat treatment It is a graph which shows the comparison of an E. coli growth curve. In the process of 3 hours, The E. coli culture has a slope of 0.2313 and then returns to the normal slope of the untreated culture. FIG. aureus growth curve and heat treatment It is a graph which shows the comparison of an aureus growth curve. In the process of 4 hours, S. The aureus culture has a slope of 0.1070 and then returns to the normal slope of the untreated culture (after 4 hours, 0.4034). FIG. 9 shows an unprocessed C.I. C. albicans growth curve and heat treatment It is a graph which shows the comparison of an albicans growth curve. C. stressed over 8 hours. albicans cultures have a slope of 0.0419. FIG. pumilus growth curve and starved B. pylori growth curve. It is a graph which shows the comparison of a pumilus growth curve. The cells experienced 7 days of nutrient deficiency at 2-8 ° C. B. Stressed in the process of 5 hours. The pumilus culture has a slope of -0.0056 and then returns to the normal slope of the untreated culture. FIG. 11 is a photomicrograph showing the results of a background test of a Schaedler blood agar medium. MILLIFLEX Rapid membrane MXHVWP124 was incubated on a Schaedler blood agar medium in a MILLIFLEX cassette for 5 days at 30-35 ° C. FIG. 11A shows a MILLIFLEX Rapid image in which the membrane was rinsed with 100 ml of fluid A. FIG. 11B shows the membrane rinsed with 100 ml of fluid D.

  The present invention relates to a method of sterility testing that is faster than conventional tests that require an incubation period of 14 days. The method can be applied to filterable liquid compositions such as liquid pharmaceutical compositions (eg, solutions, suspensions, emulsions, parenteral compositions, oral compositions, nasal compositions, ophthalmic compositions, vaccines). Particularly well suited for rapid sterility testing. In general, the method includes the step of filtering the pharmaceutical composition through a filter membrane, then transferring the filter membrane to a solid medium and allowing viable microorganisms on the membrane to grow or a sufficient amount of biomolecules, such as Incubate under appropriate growth conditions for a time sufficient to allow production of adenosine triphosphate to allow detection of the microorganism (eg 6 hours, 12 hours, 1 day, 2 days, 3 days Days, 4, 5, 6, 7).

  In one aspect, the present invention is a method for detecting viable microorganisms in a pharmaceutical composition. The method involves filtering a pharmaceutical composition (e.g., a liquid pharmaceutical composition) through a filter membrane to deposit more than one sheet of collected pharmaceutical composition and any viable microorganisms that may be present in the pharmaceutical composition. Providing a filter membrane (eg, at least three). If desired, the wash solution can then be filtered, for example, to prevent growth inhibitors, metabolic inhibitors, or detection inhibitors from being washed out of the membrane, thereby facilitating detection of microorganisms in the filtrate. The filter membrane containing the filter cake is placed in a solid medium to produce at least three filter cake cultures. The filtered culture is then applied to the filter membrane under three different conditions: aerobic conditions at 20 ° C to 25 ° C, aerobic conditions at 30 ° C to 35 ° C, and anaerobic conditions at 30 ° C to 35 ° C. The living microorganisms present are grown or cultured for a sufficient culture period to produce a sufficient amount of biomolecule, such as adenosine triphosphate, to allow detection of the microorganisms. Generally, the filtrate culture is cultured for a period of about 13 days or less, and preferably for a period substantially shorter than 13 days. The filtrate culture is then evaluated for the presence of viable microbial cells, microcolony or colonies in the membrane using any suitable method. The presence of viable microbial cells, microcolonies or colonies in the filter membrane indicates the presence of viable microorganisms in the pharmaceutical composition.

  In one aspect of the present invention, a step of providing a filterable pharmaceutical composition, a step of providing the filter membrane and providing at least three filter membranes on which the filter cake is deposited, and the three membranes in a solid medium A step of producing three filtrate cultures, culturing the first filtrate culture under aerobic conditions at 20 ° C. to 25 ° C. for a culture period of about 13 days or less; Culturing the filtrate culture at 30 ° C. to 35 ° C. under aerobic conditions, and culturing a third filter cake culture at 30 ° C. to 35 ° C. under anaerobic conditions, and in the membrane A method is provided for detecting viable microorganisms in a pharmaceutical composition comprising detecting adenosine triphosphate (ATP). The presence of ATP in the filter membrane indicates the presence of viable microorganisms in the pharmaceutical composition.

  A wide range of microorganisms can be detected using the present invention (eg, yeasts and molds, gram positive sporulating bacteria, gram negative bacteria, gram positive cocci and gram positive bacilli (both aerobic and anaerobic microorganisms)). The method can be used to detect ATCC (American Type Culture Collection) strains, and also to detect environmental microorganisms that can contaminate manufacturing equipment. For example, as described herein, the method can be referred to as Aspergillus brasiliensis ATCC 16404 (formerly known as Aspergillus nitricer ATCC 16331, Bacillus subtilis ATCC 10331, Candida albicans ATCC 10231, Clostrimus ATCC 10231, ATCC 6538, Escherichia coli ATCC 8739, Acinetobacter Iwoffi, Bacillus clausii, Bacillus idriensis, Bacillus licheni ormis, Bacillus pumilus, Bacillus sphaericus, Corynebacterium afermentans; Kocuria spez. Kocuria rhizophilia (formerly known as Micrococcus luteus), Moraxella osloensis, Penicillium spez. , Propionibacterium acnes, Staphylococcus capitis, Staphylococcus epidermidis and Staphylococcus warneri.

  Generally, the pharmaceutical composition (eg, liquid pharmaceutical composition) is filtered through a sterile filter membrane using sterilization or aseptic techniques under pressure, such as using vacuum or positive pressure. Any suitable filter membrane and filtration device can be used. Examples of suitable filter membranes are, for example, polyvinylidene fluoride membranes, glass fiber membranes, polycarbonate membranes, polyethylene terephthalate membranes, mixed cellulose esters (cellulose acetate and cellulose nitrate), phosphocellulose membranes, DEAE membranes, nylon mesh membranes. Or a polytetrafluoroethylene membrane. Preferably, the membrane is made of PVDF (polyvinylidene fluoride). Suitable filter membranes for use in the claimed invention are from about 0.1 microns (μm) to about 20 microns, from about 0.1 microns to about 15 microns, from 0.1 microns to about 12 microns,. 1 micron to about 10 microns, 0.1 microns to about 8 microns, 0.1 microns to about 6 microns, 0.1 microns to about 5 microns, 0.4 microns-12 microns, 0.4 microns-10 microns, Pore size small enough to hold microorganisms that may be present in the pharmaceutical composition, such as 0.4 micron-8 micron, 0.4 micron-6 micron, about 0.22 micron, or about 0.45 micron pore size Have Preferably, the membrane filter has a pore size of about 0.45 microns.

  Generally, at least three filter membranes containing the filter cake are prepared. This can be accomplished by filtering three separate samples of the pharmaceutical composition through three separate filter membranes. This can also be done by preparing a single filter membrane containing the pharmaceutical composition filter and cutting or splitting the filter into three parts (eg, three parts of approximately the same size) using sterilization or aseptic techniques. Can be achieved by The filter membrane containing the pharmaceutical composition filter is then placed in a suitable solid medium to produce a filter culture.

  Appropriate solid media and culture conditions can be selected to support the growth and / or metabolism of the desired microorganism to be detected. This can be accomplished, for example, by screening the media as described and exemplified herein. Preferably, the solid medium used in the present method supports the growth of a wide range of microorganisms under aerobic and anaerobic culture conditions. If desired, more than one solid medium can be used. For example, an initial medium equivalent to or better than liquid tryptic soy broth for the growth of yeast, mold and aerobic bacteria may be used; for liquid thioglycolic acid medium for the growth of anaerobic microorganisms A second solid medium equivalent to or better than the anaerobic phase can be selected; and a third equivalent to or better than the aerobic phase of the liquid thioglycolic acid medium for the growth of aerobic microorganisms Solid media can be selected. Preferably, a single solid medium is used under both aerobic and anaerobic conditions. Suitable solid media that can be used in the present invention include, for example, FTM-A (liquid thioglycolic acid medium containing 1.075% agar (final concentration)), BHI (Brain Heart Infusion Agar), Difco Brewer anaerobic. Agar medium, R2A agar medium, Schadler blood agar medium, Caso-agar medium ICR (tryptic soy agar medium), Columbia agar medium 5% blood or CDC anaerobic blood agar medium. Schaedler blood agar is a particularly preferred solid medium for use in the present method.

  Preferred solid media are suitable for supporting the growth and / or metabolism of “stressed” and “unstressed” microorganisms as described herein. Microorganisms can undergo stress, such as low or high osmotic stress, irradiation (UV light, gamma irradiation, microwaves, etc.) during process chemistry, such as during the manufacture of pharmaceutical compositions. This is desirable because it can experience ultrasound, heat, low temperature or chemical stress (eg caused by chlorine or pharmaceutical formulations).

  The filtered culture is allowed for a period of time sufficient to allow viable microorganisms on the membrane to grow to produce microcolonies or colonies, or to produce sufficient amounts of biomolecules, such as adenosine triphosphate. Incubating (ie, culturing) under appropriate growth conditions to allow detection of microorganisms. In general, one filter culture is incubated under aerobic conditions at 30 ° C. to 35 ° C. and a second filter culture is incubated under anaerobic conditions at 30 ° C. to 35 ° C. And a third filter culture is incubated at 20-25 ° C. under aerobic conditions.

  In some embodiments, the filter culture is a viable microorganism such as at least about 200 attomoles of ATP, at least about 200 femtomoles of ATP, at least about 200 picomoles of ATP, or at least about 200 nanomoles of ATP. Incubate for a period of time sufficient to allow production of detectable amounts of ATP. The culture period is about 2 to about 7 days, about 2 to about 8 days, about 2 to about 9 days, about 2 to about 10 days, about 2 to about 11 days, about 2 to about 12 days, about 2 to about It can be 13 days, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days. The incubation period for each individual filter culture can be varied appropriately and not all filter cultures need to be incubated for the same length of time. Preferred incubation times will vary based on the microorganism to be detected.

  Detection of viable microbial cells, microcolonies, or colonies present in the filter membrane after incubation of the filtrate culture using any suitable method, such as by visual inspection or preferably by using an appropriate assay. Can do. For example, a luminescent assay (eg, a luciferase assay) can be used to detect viable microbial cells, microcolonies, or colonies. Any suitable method or system may be used to detect luminescence, such as a charge coupled device camera, an image processing device, and / or image analysis software. Conveniently, image analysis software may be used to quantify or measure the number of viable microbial cells, viable microbial microcolony, or viable microbial colonies. In some embodiments, a luminescent assay such as a luciferase assay is used to detect viable microbial cells, microcolonies, or adenosine triphosphate (ATP) produced by the colonies on the filter membrane. For example, an ATP releasing reagent and a bioluminescent agent (eg, a reagent containing luciferase and luciferin) are applied to the filter membrane and luminescence occurs if viable cells produce ATP. Suitable reagents for detecting ATP by luminescence are well known in the art and are commercially available (eg, MILLIFLEX Rapid Reagent Kit; Millipore Corporation, Billerica, Massachusetts).

  Viable microbial cells, microcolonies or colonies present in the filter membrane after incubation of the filter culture are also used, eg, using a luminescent assay to detect hybridization of probes that hybridize to nucleic acids that are endogenous to the microorganism. Can be detected. The luminescence assay can include a peroxidase reaction or other suitable reaction. Suitable reagents for producing luminescence by the activity of peroxidase and other enzymes are well known and commercially available.

  Assays and detection systems that allow detection of about 10-100 yeast cells or about 1000 bacterial cells may be used. Preferably, assays and detection systems are used that allow detection of single cells or as few as about 100 cells. This can be achieved, for example, by detecting ATP produced by the microorganism. For example, using a commercially available ATP bioluminescence assay in combination with a commercially available charge coupled device camera, image processor and image analysis software (MILLIFLEX rapid microbial detection and counting system; Millipore Corporation, Billerica, Massachusetts) Can be used to detect about 200 attomoles of ATP, equivalent to about 1 yeast or mold cell or about 100 bacterial cells.

  The detection methods described herein are well suited for assessing the sterility of a filterable composition, such as a liquid pharmaceutical composition. Liquid compositions include aqueous compositions, suspensions and emulsions. The liquid pharmaceutical composition can be any liquid suitable for pharmaceutical use, including, for example, a parenteral composition, an oral composition, a nasal composition, or an ophthalmic composition. The liquid composition can be a vaccine.

  Suitable vaccines include, for example, anthrax vaccine (ANT), calmet gellan bacillus vaccine (BCG), borreliosis vaccine outer surface protein A vaccine (BORospA), diphtheria and tetanus toxoid vaccines (DT), diphtheria and tetanus toxoid and pertussis vaccines. (DTP), diphtheria and tetanus toxoid for children and acellular pertussis vaccine (DTPa), diphtheria and tetanus toxoid for adult and pertussis vaccine (DrTPar), diphtheria and tetanus toxoid and acellular pertussis And Haemophilus influenzae type b conjugate vaccine (DTPa-HIB), diphtheria toxoid and tetanus toxoid Pertussis and H. influenzae type b conjugate and poliovirus inactivated vaccine (DTPa-HIB-IPV), hepatitis A virus vaccine (HAV), hepatitis A virus and hepatitis B virus vaccine (HAV-HBV), hepatitis B Virus vaccine (HBV), Helicobacter pylori vaccine (HEL), Haemophilus influenzae type b vaccine (HIB), Haemophilus influenzae type b conjugate vaccine (HIBcn), Haemophilus influenzae type b polysaccharide vaccine (HIBps), Haemophilus influenzae type b vaccine ( Diphtheria CRRM197 protein-bound, oligosaccharide conjugated to diphtheria CRM197 toxin protein; HIB-HbOC), avian influenza (eg H5N1, H1N3) and swine influenza (eg H1N1) vaccines Including influenza virus vaccine (INF), influenza virus attenuated live vaccine (INFa), influenza virus attenuated live vaccine intranasal (INFan), influenza virus inactivated vaccine (INFi), influenza virus inactivated vaccine virion component (split virion) (INFs), influenza virus inactivated vaccine virion components A and B trivalent (INFs-AB3), influenza virus vaccine virion whole (INFw), poliovirus inactivated vaccine (IPV), meningococcus (Nyseria meningi) Tidis) vaccine (MEN), Neisseria meningitidis binding vaccine (MENcn), Neisseria meningitidis binding vaccine Serotype A, C (MENcn-AC), Neisseria meningitidis polysaccharide vaccine serotype A, C, Y, W-135 (MENps-ASYW), measles virus, epidemic ear Lower adenitis virus, rubella virus vaccine (MMR), measles virus, mumps virus, rubella virus and varicella virus vaccine (MMR-VAR), poliovirus attenuated live oral trivalent vaccine (OPV), pneumococci (Streptococcus pneumoniae) vaccine (PNU), Streptococcus pneumoniae (Streptococcus pneumoniae) combined vaccine 7 valent (PNUcn-7), Streptococcus pneumoniae (Streptococcus pneumoniae) polysaccharide 23 valent vaccine (PNUps-23), Poliovirus vaccine ( POL), rabies vaccine (RAB), rabies Human diploid cell culture (RAB-HDV), rabies vaccine purified chicken embryo cell culture (RAB-PCEC), respiratory syncytial virus vaccine (RSV), smallpox vaccine (SMA), smallpox (vaccinia virus) vaccine ( SMAvac), tetanus toxoid and diphtheria toxoid (reduced antigen dose for adults) vaccine (Td), toxoplasmosis (toxoplasm gondii) vaccine (TOX), typhoid (Salmonella typhi) vaccine (TPD), Typhoid (Salmonella typhi) vaccine attenuated live oral Ty21a strain (TPDa), typhoid (Salmonella typhi) vaccine fever and phenol inactivated dry (TPD-HP), typhoid (Salmonella typhi) vaccine Vi capsule Sugars (TPD-Vi), BCG is not a tuberculosis (Mycobacterium tuberculosis) vaccine (TUB), including varicella (varicella) (chickenpox (Chichenpox), varicella-zoster virus) vaccine (VAR).

  Suitable parenteral compositions include, for example, adenosine, alprostadil, amikacin sulfate, azithromycin, bleomycin, ceftriaxone, ciprofloxacin, cisplatin, dacarbazine, daunorubicin HCl, deferoxamine mesylate, desmopressin acetate, diltiazem, dipyridamole, doxorubicin , Enalaprilate, epirubicin, epoprostenol sodium, fluconazole, fludarabine phosphate, fludarabine phosphate, flumazenil, granisetron HCl, idarubicin HCl, ifosfamide, irinotecan HCl, leucovorin calcium, leuprolide, levocarnitine, medroxyprogesterone acetate, mesnaproate acetate Methylprednisolone, metoclopramide, mitozantrone, nalbuphine Cl, norepinephrine hydrogen tartrate, octreotide acetate, ondansetron, oxaliplatin, oxytocin, paclitaxel, disodium pamidronate, pancuronium bromide, phenylephrine bromide, phenylephrine HCl, promethazine HCl, propofol, rocuronium bromide, sulfamethoxazole , Sumatriptan succinate, tebutaline sulfate, testosterone cypionate, tobramycin, vecuronium bromide, vincristine sulfate, vinorelbine tartrate, and streptozocin sterile powder.

  Suitable oral compositions include, for example, acetaminophen and codeine phosphate tablets, acetaminophen acetazolamide tablets, acyclovir capsules, amiloride HCl, amiodarone HCl, amlodipine besylate and benazepril capsules, amlodipine besylate tablets, amoxicillin and potassium clavulanate , Amoxicillin, anagrelide, desogestrel and ethinylestradiol tablets, aspirin, atenolol, levonorgestrel and ethinylestradiol tablets, azithromycin, baclofen, benazepril HCl, benzonate, benztropine mesylate, betamethasone betamethasone benzoate betamethasone benzoate , Bicalutamide, bisopro fumarate Buproprion HCl, budesonide inhalation suspension, bumetanide, bupropion HCl, cabergoline, calcarb 600, calcitriol, calcium citrate tablets, norethindrone tablets, captopril and hydrochlorothiazide tablets, captopril, carbamazepine, carvedilol, cefadroxyl , Cephalexin, certagen, cetirizine HCl, chlordiazepoxide HCl, chlorpheniramine maleate, chlorzoxazone, cholinoid, cilostazol, cimetidine HCl, cimetidine, ciprofloxacin, ciprofloxacin, ciprofloxacin, ciprofloxacin Claris Loma Syn, clemastine fumarate, clindamycin, clomiphene citrate, clomipramine HCl, clonazepam, clotrimazole, clozapine, cromolyn sodium, cyclobenzaprine HCl, cyclosporine, cyproheptadine HCl, danazol, demeclocycline HCl, desmopressin acetate, dex Methylphenidate HCl, dextroamphetamine sulfate, diazepam, diclofenac potassium, dicloxacillin, didanosine, diltiazem HCl, diphenhydramine HCl, dipyridamole, disopyramide phosphate, divalproex sodium, dorzolamide HCl, doxazosin mesylate, maleate Carbamazepine, estazolam, estradiol, estropipe , Ethambutol HCl, ethosuximide, etodolac, famciclovir, famotidine, ferrous sulfate (ferrous sulfate), ferrous sulfate, fexofenadine HCl, finasteride, flecainide acetate, fluconazole, fludrocortisone acetate, fluocinide, fluoxetine , Flurbiprofen, flutamide, fluvoxamine, fosinopril sodium, furosemide, gabapentin, galantamine hydrobromide, gemfibrozil, glimepiride, glipizide, glucosamine sulfate, glyburide, haloperidol, hydralazine HCl, hydrochlorothiazide, hydrocodone tartrate hydrocodone Hydroxychloroquine, hydroxyurea, hydroxyzine HCl, hydroxyzine pamoate, Ndomethacin, isoniazid, ketoconazole, ketoprofen, ketorolac tromethamine, labetalol HCl, lamotrigine, lansoprazole, leflunomide, leucovorin calcium, levetiracetam, lidocaine HCl, lisinopril, loperamide HCl, lorazepam, losartan potassium, lovastatin acetic acid, prozalme acetic acid Megestrol, meloxicam, meperidine HCl, mercaptopurine, mesalamine, metformin HCl, methotrexate, methyldopa, methylprednisolone, metoclopramide, metoprolol tartrate, metronidazole, metronidazole, mexiletine, minocycline HCl, mirtazapine, misoprostol, mo epril l, mupirocin, mycophenolate mofetil, nabumetone, nadolol, naltrexone HCl, naproxen, nefazodone HCl, neomycin sulfate, niacin, nifedipine, nimodipine, nizatidine, norethindrone acetate, nortriptyline HCl, nystatin, ofloxazone, omeprazole HCl, oxaprozin, oxazepam, oxybutynin chloride, oxycodone, oxycodone HCl, pantoprazole sodium, paroxetine, penicillin V potassium, pentoxyphyllin, phenyljesic, piroxicam, pramipexole dihydrochloride, pravastatin sodium, prazosin HCl, prednisolone, malein Prochlorperazi acid , Propaphenone HCl, propoxyphene HCl, propranolol HCl, protriptyline HCl, quinapril, quinidine sulfate, ramipril, ranitidine HCl, ribavirin, risperidone, ropinirole HCl, senna-S, sennagen, silver nitrate, simvastatin, sotalol HCl, Sucralfate, tamoxifen citrate, tamsulosin HCl, terazosin HCl, terbinafine HCl, tetracycline HCl, theophylline, ticlopidine HCl, tolmetine sodium, topiramate, torsemide, tramadol HCl, trandolapril, trazodone HCl, tretinoin, ursodiol, valpro Acid, venlafaxine HCl, verapamil HCl, warfarinna Contains thorium, zaleplon, and zolpidem tartrate.

  Suitable nasal compositions include, for example, nasal sprays, antimigraine drugs, peptide drugs (hormone therapy), anesthetics, antiemetics, sedatives, azelastine hydrochloride, oxymetazoline hydrochloride, phenylephrine hydrochloride, salt Includes aqueous solution, mometasone furoate, budesonide, ipratropium bromide, and cromolyn sodium intranasal spray.

  Suitable ophthalmic compositions include, for example, lidocaine, propalacaine, tetracaine, ketorolac tromethamine ophthalmic solution, ketorolac tromethamine, naphazoline ophthalmic, brimonidine, azithromycin, bepotastine besylate, besifloxacin, betaxone, cosopt, difluprednate (Diflupredate), rotemax, ranibizumab, bimatoprost, pegaptanib, ofloxacin, desamethasone, levofloxacin, unoprostone isopropyl ophthalmic solution, cyclosporine ophthalmic emulsion, salagen, travoprost ophthalmic solution, valganciclovir HCl, viroptic, cidofovir, verteporfin, Vitrasert, vitravene, and ketotifen fumarate ophthalmic Including a liquid.

  The methods described herein can be performed using any suitable instrument or device and can be performed manually or automatically. In one preferred aspect, the method is performed using a commercially available MILLIFLEX rapid microbial detection system, which includes a sample preparation station, automatic spray station, detection tower, image analyzer, CCD camera and computer including software. (Millipore Corporation, Billerica, Massachusetts).

  In another aspect, the invention provides a sterile pharmaceutical composition (eg, vaccine, ophthalmic composition, nasal composition, oral composition, screened for viable microorganisms using the methods described herein. Parenteral composition).

Example Creating frozen collections of environmental strains from ATCC strains and production sites (contamination from surface or producer contact plates, bioburden and sterility tests) Lyophilized cultures (ATCC strains) or direct plates (environmental isolates) Microorganisms derived from were cultured in liquid tryptic soy broth or solid media (eg, Sabouraud dextrose agar) for an appropriate period of time. Genotyping of each strain was performed using MicroSeq System, Applied Biosystems. The culture was then centrifuged at 800 × G for 20-30 minutes and the pellet was resuspended in protective medium (Oxoid-CM67 with 15% glycerin). The culture was diluted and CFU (colony forming units) was checked with solid medium and filled into 2 ml Nunc Cryotube ™ vials and stored at -80 ° C. Table 1 shows a complete list of microorganisms used.

A. Stress factor studies Incubate microorganisms in diluted series parenteral formulations by applying either UV light (240-250 μW / cm ² ), heat (50-70 ° C. water bath), or for 1-10 minutes each By inducing stress, aliquots were taken every minute. Stress was applied directly to a fluid suspension of test microorganisms in the range below 100 CFU. The effect of stress was monitored by the reduction of CFU, determined using plate counting and OD measurements.

  Plate count: various effects of stress were monitored by plating stressed microorganisms on tryptic soy agar and plating each aliquot (stress was applied for 1-10 minutes, aliquots every minute Took). Results were measured after 2-7 days (depending on the strain, eg E. coli and A. niger were measured after the last 2 days, but P. acnes could not be measured before 6 days of incubation. ). The exact time required to cause a reduction of more than 50% of the initially seeded amount of CFU was measured.

OD measurement (λ = 600 nm): An overnight culture of the test microorganism is first stressed (stress parameters determined in a ≧ 50% reduction study applied) and seeded in tryptic soy broth or liquid thioglycolic acid medium (Approximately 3-4 × 10 ⁶ CFU / ml) and analyzed over a time frame of up to 8 hours (aliquots were taken to measure absorbance at each time). Incubation was performed on a shaking table (aerobic strains only). A stressed culture was compared to an unstressed culture.

Stress Factor Study 50% Reduction Results Three stress parameters were tested for each microorganism from the 22 strains. The exact parameters of each stress factor (between 1 and 10 minutes) required to cause a reduction of more than 50% in the amount of CFU initially seeded were measured. The three stress parameters tested were microbial incubation in parenteral formulations for UV light, heat and parenteral application. For example, heat causes damage to the cell membrane, RNA denatures, and this causes some cell death. UV irradiation causes mutation and termination of DNA replication (M. Strus, Rocz Panst Zakl Hig. 48 (3): 263-268 (1997)).

  The application of parenteral formulations causes chemical stress in microbial cells—the antibacterial properties of parenteral formulations have long been known. FIGS. 1-3 show examples of data obtained for Moraxella osloensis, Escherichia coli and Acinetobacter Iwoffii. For each of the 22 microbial strains, the stress parameters required to reduce the initial inoculum by ≧ 50% were found.

  In some cases, changes in colony morphology (colony grows slower but later recovers normal colony size) were observed. For example, Micrococcus luteus showed a decrease in colony size [FIGS. 4a and 4b].

  It was important not only to use a wide range of microorganisms, but also to use these microorganisms under stress for medium evaluation in rapid sterility testing. Several stress factors reduced the amount of seeded microorganisms, such as chemical stress caused by parenteral formulations and radiation stress caused by application of UV light. In contrast, heat treatment initially caused a reduction in growth rate in some of the microorganisms tested. Heat-injured cells have already been reported to take 3 to 4 hours to recover-this was confirmed in this study (M. Warsec, Appl. Microbiol., 26: 919-922 (1973)). ). Since spore-forming bacteria are broadly resistant to stress, heat stress is not useful for spore-forming bacteria and no other stress parameters are possible (P. Setlow, J. Appl. Microbiol., 101 (3): 514-525 Review (2006)). For example, B. pumilus is used as an irradiation indicator bacterium (J. Wong, PDA J Pharm Sci Technol. 58 (1): 6-14 (2004)). In the case of spore-forming bacteria, different “stress” factors were used—these were stressed by nutrient deficiencies and therefore a higher spore content was induced.

  Subsequent nutrient medium evaluation and statistical analysis revealed that the Schadler blood agar medium was the best solid medium for use in sterility testing. A t-test to analyze the difference between aerobic incubation at 20 ° C to 25 ° C and 30 ° C to 35 ° C showed that there was a significant difference between the temperatures. Since the difference was significant in 11 of the 40 cases, it is necessary to verify both incubation temperatures in a rapid sterility test.

OD measurements The stress parameters determined in the ≧ 50% reduction study were applied to each strain tested. The stressed inoculum was compared with the inoculum that was not always stressed. Microorganisms (about 3-4 × 10 ⁶ CFU / ml as the first inoculum) were incubated in either tryptic soy broth or liquid thioglycolic acid medium and incubated on a shaking table at 30 ° C. to 35 ° C. ( Aerobic strains only). Aliquots were taken at each time and the absorbance (λ = 600 nm) was measured. Data obtained for inoculum with different treatments (untreated, heat treated, treated with UV light, diluted in parenteral formulation, time / parameter used for 50% reduction experiments) is a normal plot of OD measurement data A slightly different growth curve was shown in FIG.

  By plotting the data logarithmically [FIG. The effect of heat treatment on the growth curve of E. coli is shown and makes the data independent of the amount of inoculum. The log plot has this as an advantage since it is not easy to accurately control the amount of inoculum. There is no actual difference in growth observed (depending on the parameters determined in the 50% reduction experiment) when the bacterial cultures are stressed by either UV light or parenteral formulation treatment.

  Comparison of the slopes shows that only the heat treatment (the parameter determined in the 50% reduction experiment) has an effect on the growth rate of the microorganism and that the microorganism showed real stress. UV light and dilution in parenteral formulations (both “bactericidal factors” and not “stress factors”) were not used during the media evaluation. Stress on the growth rate is best shown in detail by applying a straight line and comparing the slopes. A reduction in growth slope was shown for all microbial strains in the selected range. The examples presented herein are E.I. coli, S. et al. aureus, C.I. albicans, and B.I. pumilus. E. In the case of E. coli, the growth slope was reduced by about 3 hours, which means that the microorganism needs about 3 hours to recover [FIG. 7].

  S. aureus shows a reduction in growth slope over a 4 hour time frame [Figure 8].

  Yeast C.I. albicans showed an even longer duration of stress. The growth slope remained reduced for over 8 hours, and overnight stressed cultures restored normal growth slope [FIG. 9].

  For gram positive sporulating bacteria, heat stress is not possible. Other stress factors, UV light, and dilution in parenteral formulations did not show stress effects on the microorganism, so another stress on the spore-forming bacteria had to be found. For Bacillus and Clostridium, higher spore contents were induced in the bacterial suspension. And the OD measurement was performed [FIG. 10]. This sporulation was achieved by nutrient deprivation and by storing the overgrown microbial culture for more than 6 days at 2-8 ° C.

  All microorganisms were tested in the manner described. Difficulties were observed only in three cases. The molds Penicillium and Aspergillus grew as mycelium in liquid medium and therefore accurate OD measurement was not possible. C. Since data is available for albicans and all tested microorganisms, it is only necessary to assume that Penicillium and Aspergillus behave in the same manner. Therefore, the tested parameters are incorporated for media evaluation. In other cases, for Propionibacterium acnes (this strain grows better in FTM), the parameter for ≧ 50% reduction determined by plate count could not be reproduced in OD measurement experiments. Compared to the parameters of the ≧ 50% reduction experiment, a time lag in growth was observed only when stress was removed during a long time frame. P. acnes showed different results in OD measurements, presumably due to higher oxygen stress. An aliquot is taken every hour for OD measurement. Acnes caused certain oxygen stress. Data obtained from stress factor studies is summarized in Table 2.

Therefore, heat of stress parameters and lack of nutrients were used in the media evaluation. The selected stress factor is similar to the stress that is probably present in the production process of sterile formulations.

B. Growth Promotion Study The culture medium to be tested was inoculated with microorganisms (stressed and unstressed) in an amount of about 10-100 CFU. The experiment was performed using 5 replicate results for each incubation parameter (incubation parameters are 20 ° C to 25 ° C and 30 ° C to 35 ° C anaerobic incubation, 30 ° C to 35 ° C anaerobic incubation). Results were measured visually after 2-7 days of incubation. The raw data obtained were grouped into 6 groups for each microorganism:
1. 20 ° C to 25 ° C aerobic incubation-stressed microorganism 2. 20 ° C-25 ° C aerobic incubation-unstressed microorganism 3. 30 ° C-35 ° C aerobic incubation-stressed microorganism 4 30 ° C to 35 ° C aerobic incubation-unstressed microorganism 5. 30 ° C to 35 ° C anaerobic incubation-stressed microorganism 6. 30 ° C to 35 ° C anaerobic incubation-unstressed microorganism test The nutrient medium is grouped into subgroups.

List of solid nutrient media for pre-selection (growth promotion test with 10 unstressed strains)
FTM-A (liquid thioglycolic acid medium containing 10 g / L agar, resulting in an agar with a final concentration of 1.075%), Amimed, Allschwiil, Switzerland
・ BHI (Brain Heart Infusion Agar), γ-irradiation, hepha, Eppelheim, Germany
・ Difco Brewer Anaerobic Agar, γ-irradiation, hepha, Eppelheim, Germany
・ R2A agar, Oxoid, Great Britain
・ Schaedler blood agar medium, gamma irradiation, hepha, Eppelheim, Germany
Caso-agar medium ICR (tryptic soy agar medium), γ-irradiation, hepha, Eppelheim, Germany
・ Columbia agar 5% blood, BioMerieux, France
CDC anaerobic blood agar medium, gamma irradiation, hepha, Eppelheim, Germany.

List of solid nutrient media for final study (growth promotion test with 22 strains in unstressed and stressed state)
・ Difco Brewer Anaerobic Agar, γ-irradiation, hepha, Eppelheim, Germany
・ Schaedler blood agar medium, gamma irradiation, hepha, Eppelheim, Germany
Caso-agar medium ICR (tryptic soy agar medium), γ-irradiation, hepha, Eppelheim, Germany
CDC anaerobic blood agar medium, gamma irradiation, hepha, Eppelheim, Germany
All γ-irradiated media was supplemented to maintain the irradiation process.

Nutrient Medium Evaluation Nutrient medium evaluation studies were conducted in two parts: initial pre-selection (proliferation promotion test with 10 unstressed strains, test with 8 agar media) into 4 media. Reduced, which meant that only these media were considered more closely. FTM-A agar, brain heart infusion agar, R2A agar and Columbia agar 5% blood were excluded in this preliminary selection.

  In evaluating nutrient media, the following four media were tested in detail: tryptic soy agar, CDC anaerobic blood agar, Schadler blood agar, and Difco Brewer anaerobic agar. The data obtained were grouped and statistically analyzed using ANOVA for each of the 22 strains seeded in both stressed and unstressed conditions. Three incubation parameters, 20 ° C-25 ° C and 30 ° C-35 ° C aerobic incubation and 30 ° C-35 ° C anaerobic incubation, and two different stress states for each microorganism (stressed and unstressed) To form 6 groups for each microorganism (x22 strains). ANOVA for each of these groups was performed and credits for good growth promoting properties were summarized for 22 microorganisms on each agar medium. If it achieved the highest count, 1 credit was given to the group / agar. The group / agar medium that was not significantly different from this highest count agar medium also received 1 credit.

  In the 20 ° C. to 25 ° C. aerobic incubation group, the Schadler blood agar collected 30 credits, the CDC anaerobic blood agar collected 27 credits, the tryptic soy agar medium collected 24, and the Difco Brewer anaerobic agar collected 17 credits. [Tables 3A and 3B]. P. acnes and Cl. Sporegenes did not grow aerobically and therefore did not collect credits. Stressed A. niger, B.M. pumilis, B. et al. sphaericus and B. et al. Idriensis was 0 credits due to the low count (0-5 CFU) in all tested agar media.

In the 30 ° C.-35 ° C. aerobic incubation group, the Schaedler blood agar collected 28 credits, the tryptic soy agar collected 28 credits, the CDC anaerobic blood agar 27 credits, and the Difco Brewer anaerobic agar 24 Credits were collected [Tables 4A and 4B]. P. acnes and Cl. Sporegenes did not grow aerobically and therefore did not collect credits. B. Stressed idriensis and M.I. osloensis was 0 credits due to the low count (0-5 CFU).

In the 30 ° C-35 ° C anaerobic incubation group, CDC anaerobic blood agar collected 25 credits, Schadler blood agar 25 credits, tryptic soy agar 21 credits, and Difco Brewer anaerobic agar 17 credits. Collected [Tables 5A and 5B]. B. Stressed Idriensis was 0 credits due to the low count (0-5 CFU).

These data indicate that the Schedler blood agar has the best growth promoting properties in an aerobic condition and that the Schedler blood agar obtained the same amount of credit as the CDC anaerobic blood agar in anaerobic conditions. Is shown. Therefore, Schaedler blood agar was selected as the appropriate agar for the 22 test strains and for the three tested incubation parameters. The composition of the Schaedler blood agar medium (13) (modified by hepha, Eppelheim, Germany) is as follows: 10 g casein peptone, 1 g soy flour peptone, 2 g meat peptone, 1 g meat extract (exact), yeast extract 5 g of product, glucose 2 g, NaCl 5 g, dipotassium hydrogen phosphate 2.5 g, agar bacteriological grade 14 g, sheep blood 50 ml, amino acid, buffer, hemin, vitamin K, gamma irradiation: 9-20 kGy.

Differences between aerobic incubations at 20 ° C to 25 ° C and 30 ° C to 35 ° C to show whether there is a significant difference between incubation temperatures 20 ° C to 25 ° C and 30 ° C to 35 ° C (aerobic incubation) In addition, a t-test was performed using the data from the nutrient medium evaluation.

  A t-test was performed on 40 groups consisting of all aerobic microorganisms (except Cl.sporegees and P.acnes) both in the stressed and unstressed state. Significant differences occurred in 11 out of 40 groups. Compared to the 20 ° C. to 25 ° C. group, the incubation parameters 30 ° C. to 35 ° C. showed 5 times higher amounts of colony forming units (derived from the same inoculum). The 20 ° C. to 25 ° C. group produced higher values in 6 cases. The t-test showed that there was a significant difference between the temperatures. Since the difference was significant in 11 of the 40 cases, it is necessary to verify both incubation temperatures in a rapid sterility test. These results highlight the importance of both incubation temperatures, not only for the conventional sterility test, but also for this rapid sterility test.

Statistical analysis of data For the statistical analysis of raw data, the following method implemented in Minitab (R) Release 14.20 Statistical Software was used.

  ANOVA (Analysis of Variance) compares the mean values. ANOVA is similar to regression analysis because it is used to investigate and model the relationship between reaction variables and one or more independent variables (between groups). It uses hypothesis testing, which means testing the null hypothesis. ANOVA produces a p-value. The p-value represents the possibility of a type 1 error, or negates the null hypothesis if it is true. The cutoff value is 0.05-if the p-value is lower than 0.05, corresponding to a 95% confidence limit, the null hypothesis is denied.

The requirements for using ANOVA analysis are:
Normal distribution of 4 subgroups (nutrient medium), p value ≧ 0.05
• Equal variance test using Bartlett's Test: p value ≧ 0.05, and • Leavene's Test: p value ≧ 0.05.

An ANOVA p-value ≧ 0.05 means that there is no significant difference between the subgroup / agar medium, and a p-value <0.05 means that there is a significant difference between the subgroup / agar medium. Means. All agar media that showed no significant difference to the highest average value (including those with the highest average value) were rated as 1 credit. The agar medium that showed a significant difference with respect to the highest average value was not credited. Outlier analysis was performed using Dixon's Test (WJ Dixon, Ann. Math. Statist. 21: 488-506 (1950); WJ Dixon, Biometrics. 9: 74-89). 1953); USP <1010> “Analytical data-interpretation and treatment”, Pharmaceutical Cooperative Forum, USP30-NF, Second Supplement The United States Pharmacopoeia. To this, the N values including a set of observations during the test were placed in ascending _{order: X 1 <X 2 <···} <X N. The statistical experiment Q value (Qexp) was then calculated. This is the ratio defined as the difference of the suspicious value from its closest value divided by the range of suspicious values (Q: rejection quotient). Therefore, the following Qexp values were used to test x ₁ or X _N (as potential outliers):
The resulting Qexp value was compared to the rejection Q value (Qcrit) found in the table. If Qexp> Qcrit, the suspicious value can be characterized as an outlier and rejected. If not, the suspicious value must be retained and used in all subsequent calculations. Other ways in which ANOVA can be performed are: Subgroups (four nutrient media within groups) that do not follow a normal distribution and are significantly lower than the other subgroups, they are inappropriate for ANOVA Therefore, it was excluded. If the CFU value of the subgroup within the group was too low (0-5 CFU), it gave a final result of 4 times 0 credits (4 times 0 credits). In all other cases, retests had to be performed. A t-test (also using Minitab® Release 14.20 Statistical Software) is used to compare the mean of two samples; the t-test is associated with two variations in the data. Compare the actual difference between the mean values (expressed as the standard deviation of the difference between the mean values).

C. General Description of Millipore Milliflex® Rapid Microbiology Detection System A sample under test (diluted in 100 ml of rinsing fluid to ensure proper distribution of microorganisms in the membrane) was added to Milliflex (Miflflex). ) (R) PLUS pumps are used to filter through Milliflex (R) Rapid filters, and potential contaminants are trapped in the filter membrane (pore size: 0.45 [mu] m).

  After filtration, the filter is applied to the solid nutrient medium using a Milliflex® system, where the sterile Milliflex® filter snaps on the Milliflex® cassette and the funnel is released. . Due to the Milliflex® system that moves the membrane to the nutrient medium, there is little or no risk of secondary contamination due to membrane handling. Since the cassette is tightly closed, the risk of secondary contamination during incubation is low. By incubating the filter with a Milliflex® cassette, growth of potential contaminant (s) into microcolonies can occur. At the end of the incubation period, the filter is separated from the Milliflex® cassette and applied to an Autospray Station in a layered flow hood. The microcolony must have a certain size (about 10-100 yeast cells, 1000 bacterial cells) for detection in Milliflex® Rapid, so the post-incubation step is secondary It is not an important process for mechanical mixing. If an environmental or operator-derived microorganism joins the membrane during the post-incubation step, it will not be detected due to the lack of incubation and thus the lack of a certain amount of ATP required for detection. .

  The AutoSpray Station sprays the membrane with an ATP releasing agent and then a bioluminescent reagent.

  The reaction chemistry shown here is the basis for Milliflex® Rapid detection (WD McElloy, Adv. Enzymol. Relat. Areas. Mol. Biol. 25: 119-166 (1963)).

Luciferin + luciferase, Mg ²⁺ → oxyluciferin + AMP + hv (λ = 562 nm)

(Mg ²⁺ is magnesium, ATP is adenosine triphosphate, AMP is adenosine monophosphate, hv is emitted photon, and λ is wavelength).

  In order to capture the maximum number of emitted photons, a rapid transfer of the sprayed membrane from the Autospray Station to the Milliflex® Rapid Detection Tower is necessary. The possible light signal is detected with a CCD camera (charge coupled device) and an algorithm of Milliflex®, Rapid software calculates the amount of microcolony.

No bioluminescence background in Milliflex® Rapid detection The Milliflex® Rapid Microbiology Detection System uses a Schaedler blood agar medium to determine whether this medium produces a bioluminescent background. A test was conducted. For this, the media cassettes were incubated with MXHVWP124 membrane (polyvinylidene fluoride membrane, pore size 0.45 μm) rinsed with either 100 ml of fluid A or fluid D. The incubation temperature was 30 ° C. to 35 ° C., and the incubation was performed for 5 days. FIG. 11 shows that there is no bioluminescent background from the Schaedler blood agar, meaning that there is no interfering ATP remaining in the gamma irradiated nutrient medium. Schaedler blood agar is suitable for use in the Milliflex® Rapid System.

  The entire teachings of all documents cited herein are hereby incorporated herein by reference.

Claims (21)

A method for sterility testing of a liquid pharmaceutical composition comprising:
a) providing a filterable pharmaceutical composition;
b) filtering the pharmaceutical composition to provide at least three filter membranes on which the filter cake of the pharmaceutical composition is deposited;
c) placing the at least three filter membranes in a solid medium to produce at least three filtrate cultures;
d)
i) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions;
ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and
iii) at least one filter cake culture at 30 ° C. to 35 ° C. under anaerobic conditions,
Culturing, wherein none of the filter cultures are cultured for a period longer than 13 days; and e) detecting viable microbial cells, microcolony or colonies in the filter membrane, Wherein the presence of viable microbial cells, microcolonies or colonies in the filter membrane indicates the presence of viable microorganisms in the pharmaceutical composition.   The method of claim 1, wherein step b) further comprises filtering the wash solution after filtering the pharmaceutical composition. The filter membrane is a polyvinylidene fluoride membrane, glass fiber membrane, polycarbonate membrane, polyethylene terephthalate membrane, mixed cellulose ester (cellulose acetate and cellulose nitrate), phosphocellulose membrane, DEAE membrane, nylon mesh membrane, or polytetrafluoroethylene ( 3. The method according to claim 1 or 2, wherein the membrane is a polytetrafluoroethylene) membrane. The method according to claim 1, wherein the filter membrane has a pore size of 0.45 μm.   The solid medium is FTM-A (liquid thioglycolic acid medium (final concentration) containing 1.075% agar), BHI (brain heart infusion agar medium), Difco brewer anaerobic agar medium, R2A agar medium. The Schedler blood agar medium, Caso-agar medium ICR (tryptic soy agar medium), Columbia agar medium 5% blood, and CDC anaerobic blood agar medium. The method described in 1.   6. The method according to any one of claims 1 to 5, wherein in step d), the filter cake culture is cultured for a period sufficient to produce a detectable amount of ATP.   The method according to claim 6, wherein the filtered culture is cultured for a period of 2 to 7 days.   The method according to any one of claims 1 to 7, wherein in step e), viable microbial cells, microcolonies or colonies are detected using a luminescence assay. 9. The method of claim 8, wherein the luminescent assay detects viable microbial cells, microcolony, or adenosine triphosphate (ATP) produced by the colony in the filter membrane.   9. The method of claim 8, wherein the luminescence assay comprises a luciferase assay.   9. The method of claim 8, wherein the luminescence assay detects a nucleic acid hybridization product formed between a probe and a nucleic acid that is endogenous to the microorganism.   The method of claim 11, wherein the luminescence assay comprises a peroxidase reaction.   13. A method according to any one of claims 8 to 12, wherein luminescence is detected using a charge coupled device camera and image analysis software.   14. A method according to any one of claims 8 to 13 wherein the viable microbial cells, viable microbial microcolonies, or viable microbial colonies are counted.   The method according to any one of claims 1 to 14, wherein the pharmaceutical composition is a liquid composition.   16. The method of claim 15, wherein the liquid composition is a parenteral composition, an oral composition, a nasal composition, or an ophthalmic composition.   The method of claim 15, wherein the liquid composition is a vaccine.   The vaccine is an anthrax vaccine; tuberculosis vaccine; borreliosis vaccine; diphtheria toxoid and tetanus toxoid vaccine; diphtheria toxoid and tetanus toxoid and pertussis vaccine; diphtheria toxoid and tetanus toxoid and acellular pertussis vaccine; Acellular pertussis and Haemophilus influenzae type b conjugate vaccine; Diphtheria and tetanus toxoid and acellular pertussis and Haemophilus influenzae type b conjugate and poliovirus inactivated vaccine; Hepatitis A virus vaccine; Hepatitis A virus and Hepatitis B virus Vaccine; Hepatitis B virus vaccine; Helicobacter pylori vaccine; Haemophilus influenzae type b vaccine; Influenza virus vaccine Poliovirus vaccine; Neisseria meningitidis vaccine; measles virus, mumps virus, rubella virus vaccine; measles virus, mumps virus, rubella virus and chickenpox Viral vaccine; Streptococcus pneumoniae vaccine; Rabies vaccine; Respiratory syncytial virus vaccine; Smallpox vaccine; Toxoplasmosis (toxoplasm gondi) vaccine; Typhoid fever (Salmonella typhi) vaccine; Tuberculosis (Myco) 18. The method of claim 17, wherein the method is selected from the group consisting of (bacteria tuberculosis) vaccine; and varicella (varicella, varicella-zoster virus) vaccine.   The method according to claim 17, wherein the vaccine is a bird flu vaccine or a swine flu vaccine. A method for sterility testing of a liquid pharmaceutical composition comprising:
a) providing a filterable pharmaceutical composition;
b) filtering the pharmaceutical composition to provide at least three filter membranes on which the filter cake of the pharmaceutical composition is deposited;
the at c) said at spaced three filters over main Nburan to solid medium, the step of producing at least three filtrates Tobutsu culture;
d)
i) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions;
ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and iii) at least one filter culture at 30 ° C. to 35 ° C. under anaerobic conditions;
Culturing, wherein none of the filter cultures are cultured for longer than 13 days; and e) detecting adenosine triphosphate (ATP) in the filter membrane, Wherein the presence of ATP in the filter membrane indicates the presence of viable microorganisms in the pharmaceutical composition. A method for preparing a sterile pharmaceutical composition, wherein the pharmaceutical composition is screened for viable microorganisms and has the following sterility:
a) providing a filterable pharmaceutical composition;
b) filtering the pharmaceutical composition to provide at least three filter membranes on which the filter cake of the pharmaceutical composition is deposited;
c) placing the at least three filter membranes in a solid medium to produce at least three filtrate cultures;
d)
i) at least one filter culture at 20 ° C. to 25 ° C. under aerobic conditions;
ii) at least one filter culture at 30 ° C. to 35 ° C. under aerobic conditions; and
iii) at least one filter cake culture at 30 ° C. to 35 ° C. under anaerobic conditions,
A step of culturing, wherein none of the filtrate cultures are cultured for a period longer than 13 days; and e)
(I) survival in the filter membrane microbial cells, comprising the steps of detecting the microcolonies or colonies, where viable microbial cells in the filter membrane, the presence of microcolonies or colonies for the presence of viable microorganisms in the pharmaceutical composition shown, process or a step of detecting (ii) adenosine triphosphate in said filter membrane (ATP),, the presence of ATP in the filter membrane herein indicates the presence of viable microorganisms of the pharmaceutical composition, A method identified using a method for detecting viable microorganisms in the pharmaceutical composition comprising a step.